The etiologies of aging, inflammation and neurodegenerative diseases, have all (with varying degrees of certitude) been associated with increased exposure to reactive oxygen species (ROS) and/or reactive nitrogen species (RNS). We have developed a cell-based model of nitrosative stress and showed that protein disulfide isomerase (PDI) is negatively regulated by S-glutathionylation. PDI is the most abundant protein in the endoplasmic reticulum (ER) and facilitates the protein folding process. Three signaling pathways collectively referred to as the unfolded protein response (UPR) have evolved to ensure that misfolded or unfolded proteins do not accumulate in the ER. Excess or prolonged ER stress leads to apoptosis. As such, ER stress and activation of the UPR are associated with a wide variety of disease pathologies. Our current understanding of the UPR begins with sensing the accumulation of misfolded proteins. The cellular malfunction that directly leads to impairment of the protein folding capacity of the cell is not yet fully defined. The central hypothesis is that Sglutathionylation of PDI contributes to, and may even be sufficient for, the induction of ER stress and may concomitantly lead to apoptosis. Four aims are proposed to 1) Identify target cysteine residues in PDI that are S-glutathionylated following oxidative and nitrosative stress;2) Determine the consequence of S-glutathionylation on PDI structure/function;3) Elucidate the role of Sglutathionylation of PDI in the UPR;and 4) To determine the inherent changes in PDI and progressive loss of dopamine neurons in drug-induced and genetic models of PD and evaluate the therapeutic efficacy of NOV-002, a glutathione disulfide mimetic. The connection between S-glutathionylation of PDI and initiation of this cascade is important. There is compelling evidence suggesting that ER-stress contributes to neuronal cell death and ultimately the clinical manifestations of neurodegenerative diseases. Neuronal cells have an enriched ER and will provide an excellent model to study both ER stress and to define molecular targets of ROS/RNS.